In this proposal we optimize an influenza neutralization assay for easy identification of a broad spectrum of antiviral compounds. We have modified the 'gold-standard' neutralization assay so that it can be used in high-throughput screening of chemical libraries. Wells that contain a monolayer of cells are exposed to a small amount of influenza in the absence or presence of compounds that are being screened. Viral strains A/PR/8/34 (H1N1) and B/HK/73 will be used following BSL2 practices to develop and optimize this assay. We have modified the neutralization assay and reduced the time needed to complete analysis by measuring viral neuraminidase (NA) activity to quantitate the amount of virus present in the well. NA activity is measured within 16 hr of assay start after the addition of a small NA substrate that has a fluorescent product. This assay is therefore fast and sensitive. It has additional advantages over assays that have a single biochemical target: (1) it is based on a well-established neutralization assay that is routinely used as a gold standard for titration of antisera; (2) it can identify compounds that inhibit virus replication at any point of the life cycle. Both NA and M2 inhibitors are used as controls. It is likely that a broad range of antivirals with different mechanisms of action will be identified using this assay; (3) it is easily adapted to all types and subtypes of influenza viruses as the read out (NA enzyme activity) is not strain specific; (4) it is sensitive, with accurate measurement of the inhibitory dose of a compound; (5) it is reproducible - obtaining consistent results within assay replicates as well as assay repeats; (6) it is adaptable to automated liquid handling and screening systems. We will optimize the assay in 96 and 384-well plate formats, validate the assay using automated liquid handling and screening systems and establish the parameters required to identify compounds that specifically inhibit influenza virus replication. [unreadable] [unreadable] [unreadable] [unreadable]